Determination of binding curves via protein micropatterning in vitro and in living cells

Abstract

Quantification of protein interactions in living cells is of key relevance for understanding cellular signaling. With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatterning as a convenient and versatile method for such investigations. Cells are grown on surfaces containing micropatterns of capture antibody to a bait protein, so that the bait gets rearranged in the live cell plasma membrane. Upon interaction with the bait, the fluorescent prey follows the micropatterns, which can be readout with fluorescence microscopy. In this study, we addressed the interaction between Lck and CD4, two central proteins in early T-cell signaling. Binding curves were recorded using the natural fluctuations in the Lck expression levels. Surprisingly, the binding was not saturable up to the highest Lck expression levels: on average, a single CD4 molecule recruited more than nine Lck molecules. We discuss the data in view of protein-and lipid-mediated interactions. '

Protein micropatterns printed on glass: Novel tools for protein‐ligand binding assays in live cells

“…). This fits with the pattern element sizes between 3 and 10 μm given in the literature for similar approaches . For even spreading, treatment of the interspaces with a protein ( e.g.…”

Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane

“…To this end, we developed a surface architecture for spatial organization of transmembrane receptors within the plasma membrane of live cells. Capturing of transmembrane receptors in living cells by patterning ligands or antibodies on solid support has been pioneered and was applied very successfully for studying spatial regulation of signaling processes (Torres et al, 2008a,b; Salaita et al, 2010; Xu et al, 2011; Gandor et al, 2013) and the assembly of signaling complexes (Schwarzenbacher et al, 2008; Arrabito et al, 2013; Sunzenauer et al, 2013; Lanzerstorfer et al, 2014) in a systematic and quantitative manner. Here, we have expanded this concept toward a generic methodology for quantitative interaction analysis with bait proteins micropatterned within the plasma membrane of living cells.…”

Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells

“…Recently, capturing of proteins in the plasma membrane of living cells by micropatterning ligands or antibodies on solid support has been introduced as a novel tool for controlling spatial organization of proteins in the plasma membrane . These techniques have been successfully applied for unraveling the role of spatial regulation in signaling processes, but also for monitoring the assembly of signaling complexes as well as interactions of downstream effector proteins in a systematic and quantitative manner . However, a fundamental drawback of currently available approaches is that protein capturing already occurs during adhering and culturing cells on surface‐functionalized substrates, thus potentially altering cellular homoeostasis or even activating cellular signaling …”